Protective Mechanism of Gandou Decoction in a Copper-Laden Hepatolenticular Degeneration Model: In Vitro Pharmacology and Cell Metabolomics

Gandou decoction (GDD) is a classic prescription for the treatment of hepatolenticular degeneration (HLD) in China; however, the liver-protecting mechanism of this prescription needs further evaluation. In the present study, we explored the protective mechanisms of GDD in a copper-laden HLD model using integrated pharmacology and cellular metabolomics in vitro. The results revealed that GDD could significantly promote copper excretion in copper-laden HLD model cells and improve the ultrastructural changes in hepatocytes. In addition, GDD could decrease the extent of lipid peroxidation, levels of reactive oxygen species, and the release rate of lactate dehydrogenase while increasing the activity of superoxide dismutase and the ratio of glutathione to oxidized glutathione in the copper-laden HLD model cells. On conducting statistical analysis of significant metabolic changes, 47 biomarkers and 30 related metabolic pathways were screened as pharmacological reactions induced by GDD in HLD model cells. d-glutamate and d-glutamine metabolic pathways showed the highest importance and significance among the 30 metabolic pathways, and the differential expression levels of the glutamine synthetase (GS) and the renal type and liver type GLS (GLS1 and GLS2) proteins were verified by Western blotting. Collectively, our data established the underlying mechanism of GDD therapy, such as the promotion of copper excretion and improvement in oxidative stress by regulating the expressions of GS, GLS1, and GLS2 protein to protect hepatocytes from injury.


Establishment and Evaluation of HLD Hepatocyte Model
HLD hepatocyte model was established by RNA interference. The experiment was divided into 6 groups: control group, shNC group, shATP7B-1 group, shATP7B-2 group, shATP7B-3 group and shATP7B-4 group. After washed the 6-well plate with serum-free medium twice, the transfection complex of plasmid DNA-Lipofectamine TM 2000 was added to the cells. Meanwhile, the serum-free medium without transfection reagent was added to the control group. After incubation for 6 hours, the control medium was replaced and cultured for 48 hours. Follow-up experiments were carried out.

Determination of Transfection Efficiency
After 48 hours, the nucleus was stained with DAPI, and the cell transfection was observed under a confocal laser microscope. The transfection efficiency was the ratio of the number between green fluorescent cells and blue fluorescent cells.

Real-Time PCR (qPCR)
The total RNA of cells in each group was extracted by the Trizol method and then reversely transcribed into cDNA. Then PCR amplification was carried out with the internal reference of β-actin. 10 μL system (including 5 μL of 2×SYBR Green mixture, 1 μL of 10 μM ATP7B upstream primer and downstream primer, 1 μL of cDNA and 2 μL of RNase Free water) was pre-denatured at 95 ℃ for 1 min, then the 40 cycles were operated as follows: denaturation at 95 ℃ for 20 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 1 min. Finally, it was extended for 5 min at 72 ℃. Each sample was provided with 3 multiple holes. The experimental results are expressed by the relative expression of the target gene calculated by the 2 -△ △ Ct method. PCR primers were synthesized by GenePharma Co. Ltd. (Shanghai, China). All primers were as follows: ATP7B, Forward 5'-CTGGAGAGCTCCATCCTGAG--3', Reverse 5'-CAGGCAGAGAACAACAGACG-3', β-actin, Forward 5'-GAGCGCAAGTACTCTGTGTG-3', Reverse 5'-CCTGCTTGCTGATCCACATC-3'.

Western Blot Assay
The protein isolated from the cell was electrically transferred to a methanolactivated PVDF membrane after separation by 10 % SDS-PAGE. After being blocked by a TBST buffer containing 5 % skim milk for 2 hours, it was incubated overnight at 4 ℃ with the corresponding first antibody diluent (mouse anti-ATP7B antibody and rabbit anti-β-actin antibody with a dilution ratio of 1: 200). Then, the samples were washed with TBST 3 times, each time for 10 min, and were incubated with HRP labeled goat anti-mouse lgG and HRP labeled goat anti-rabbit lgG (The dilution ratio of them were 1: 10000) for 2 hours. After washed the film, the bands were displayed with an ECL luminescence kit, and each sample was repeated 3 times.

Evaluation of Hepatolenticular Degeneration Hepatocyte Model
As shown in the figure, there was no green fluorescence expression of GFP in the control group ( Figure S1A), but the green fluorescence-labeled by GFP could be seen in shNC group and four shATP7B groups (Figure S1B-F) after 48 hours transfection. The transfection efficiency of the shATP7B-4 group ( Figure S1F) was the highest and it was about 70%. RT-PCR results (Figure S1G-K) confirmed that compared with the control group, the ATP7B mRNA expression levels of shATP7B-1, shATP7B-2, shATP7B-3, and shATP7B-4 were down-regulated to 35.0% ± 5.1%, 30.5% ± 3.1%, 36.8% ± 4.8% and 29.5% ± 3.9%. Especially in the shATP7B-4 group, the gene interference effect is the strongest. At the same time, western blot results ( Figure S1L) showed that the expression level of ATP7B protein in the plasmid transfection group was significantly down-regulated (p <0.01), and shATP7B-1, shATP7B-2, shATP7B-3 and shATP7B-4 were down-regulated to 37.2% ± 3.4%, 32.6% ± 5.1%, 34.3% ± 6.0% and 30.5% ± 2.8% respectively. In summary, the shATP7B-4 group has the strongest transfection effect and can be used for the establishment of copper-loaded HLD hepatocyte model.

The Method Validation of Cell Metabolomics
Under two separation systems, a characteristic ion peak (Rt-m/z HILIC column: negative ion 3.99 min-237.0911, positive ion 4.55 min-754.1806, C18 column: negative ion 2.68 min-391.2828, positive ion 2.92 min-532.3852) was randomly selected from each QC sample in dual ESI mode for UPLC-MS methodology verification ( Table S2). The instrument precision results of Q-TOF/MS by hydrophilic column separation showed that the relative standard deviations (RSD) of retention time and peak area were below 0.16% and 6.81%, respectively. Similarly, the RSD results of the reverse chromatographic separation system were inferior to 0.35% and 5.05%. In addition, the sample stability results showed in the HILIC system that RSD of retention time and peak area were under 0.24% and 7.97%, and in the C18 system that were under 0.31% and 6.65%. The overlapping spectral analysis of QC samples proved that the instrument 3 was stable and the metabolomics analysis method was reliable. Figure S1.